A new mycotoxin, sambutoxin (1), has been isolated from wheat culture of Fusarium sambucinum which was obtained from rotted potato tuber. Its chemical structure was elucidated as 3-{5-methyl-6-[(E)-1,3,5-trimethylhept-1-enyl]tetrahydropyranyl}-5-(p-hydroxyphenyl)-1-methyl-4-hydroxy-2(1H)-pyridone mainly by NMR analyses. Fusarium species are spread over the world and produce toxic metabolites belonging to various chemical groups (e.g. trichothecenes, zearalenone, fumonisins, etc.). Fusarium mycotoxins have caused numerous mycotoxicoses in humans and farm animals such as alimentary toxic aleukia (ATA) in the Soviet Union, moldy corn toxicosis in the midwestern United States, red mold disease in Japan, and moldy barley toxicosis in Korea. These mycotoxicoses were described as food refusal, vomiting, weight loss, congestion of and hemorrhage in tissues, and death in animals and/or humans. Although a number of studies have been carried out to identify mycotoxins responsible for the hemorrhagic syndromes, only a small portion of them have been attributed to T-2 and diacetoxyscirpenol. In the course of screening of toxic metabolites from Fusarium species, we purified a new hemorrhagic mycotoxin, sambutoxin (1), from an isolate of F. sambucinum PZF-4. In this paper, we describe the isolation and the structure elucidation of 1. The fungus was isolated from rotted potato tuber in Korea. It was incubated on wheat culture for 2 weeks at 25°C followed by 2 weeks at 10°C. The toxin was extracted and chromatographed successively on the following columns with solvents as indicated: Silica gel (70-230 mesh), CHCl3 to CHCl3-CH3OH (9:1); Silica gel (230-400 mesh), EtOAc-n-Hexane (3:1); Prep-HPLC (ODS column), CH3OH-H2O (4:1). Recrystallization from methanol gave colorless and prismatic crystals; UV (CH3OH) λmax 213 nm (38000), 233 nm (17000), 254 nm (29000); m.p., 196.5-197.5°C; [α]D 25 -200° (C=0.1 CH3OH). High-resolution FAB mass spectrometry gave the molecular formula C28H40NO4 (MH+, calcd, m/z 454.2959; found, m/z 454.2957). The IR absorptions (KBr) at ν 3185 cm-1, and 1650 and 1560 cm-1 suggested the presence of hydroxy and tertiary amide groups, respectively. All 1H- and 13C-NMR chemical shifts together with long-range 1H-13C correlations observed in the HMBC spectrum for 1 are presented in Table 1. The p-hydroxyphenyl group (A) was deduced from the typical A2M2 two doublets in the 1H-NMR spectrum and two 13C signals from the chemically equivalent pairs of carbons C2', C6' and C3', C5' in the 13C-NMR spectrum. The structure of hydroxypyridone (B) was mainly determined by chemical shifts of carbons and HMBC spectral data. Cross peaks due to 2/3JCH between the C4-OH proton and C3, C4, and C5 in the HMBC spectrum and the chemical shifts of the carbons clarified the partial structure C3 through C5 and the position of hydroxy group. The chemical shift (δC 161.4 ppm) of C2 showed it to be the carbon of the amide group. HMBC spectra clarified the connectivities around the amide by giving cross peaks due to 2/3JCH couplings between H6/C5, H6/C23, H6/C2, H23/C6, and H23/C2. The proton spin system of H7-H11 (including H22) of pyranyl ring (C) was determined by 1H-1H COSY, 1H-1H decoupling, and 1D HOHAHA experiments. The ether linkage of C between C7 and C11 was clarified by NOE between H7/H11. The relative stereochemistry of the ring was established by 1H-NMR coupling constants and NOE experiments. H7 was assigned as axial on the basis of a large coupling with H8a (Jv,a=11.3 Hz). NOEs between H7/H9a and H7/H11 revealed that H9a and H11 also are axial. The strong coupling between H9a and H10 (J9a,10=12.7 Hz) showed that H10 was axial and that the C22 methyl group was equatorial. The proton spin system of H13-H20 of trimethylheptenyl moiety (D) was determined by 1H-1H COSY, 1H-1H decoupling, and 1D HOHAHA experiments. The positions of C12 and C21 were clarified by HMBC cross peaks between H14/C12, H21/C12, H21/C13, and H13/C21. The E geometry of the C12 and C13 double bond was established by NOEs between H11/H13 and H21/H14. The connectivities between A, B, and C were determined by HMBC cross peaks between H6/C1', H7/C2, H7/C3, and H7/C4, and 1H-13C COLOC cross peaks between H6/C1', H2'(H6')/C5, and H7/C3. HMBC also clarified the connectivity between C and D by giving cross peaks due to 3JCH couplings between H11/C13, H13/C11, H21/C11, and H11/C21. Thus, the chemical structure of 1 was elucidated as 3-{5-methyl-6-[(E)-1,3,5-trimethylhept-1-enyl]tetrahydropyranyl}-5-(p-hydroxyphenyl)-1-methyl-4-hydroxy-2(1H)-pyridone. The stereochemistry at C14 and C16 is not established yet. X-ray crystallography studies to determine the absolute stereochemistry are currently in progress. As fungal metabolites containing 1-methyl-4-hydroxy-2(1H)-pyridone or its homologous moieties, several compounds from some genera have been isolated, namely funiculosin, tenellin, bassianin, ilicicolin H, harzianopyridone, leporin A, and fischerin. The purified 1 caused toxic effects in rats including body weight loss, hemorrhages in the stomach and intestines, and finally death when rats were fed the diets supplemented with 0.05 and 0.1% sambutoxin. The toxin was also toxic to chick embryos and the 50% lethal concentration was 29.6 μg/egg. Sambutoxin (1) is a new type of mycotoxin having hydroxyphenylpyridone and pyranyl moieties. It was also produced by F. oxysporum isolates obtained from rotted potato tubers. This is the first report on the production of bioactive secondary metabolite containing phenylpyridone and pyranyl moieties by Fusarium species.